靶向腸道菌群治療胃腸疾病研究進展

邵好青,譚周進

邵好青,譚周進,湖南中醫(yī)藥大學 湖南省長沙市 410208

0 引言

近年來,腸道微生態(tài)的研究如火如荼,成為多種腸道及腸外疾病的研究熱點.腸道微生物群本質(zhì)上是一種為宿主提供代謝、免疫和保護功能的微生物器官,微生物之間及與宿主之間的相互作用在維持腸道正常生理功能、維護腸上皮屏障、調(diào)節(jié)機體免疫以及拮抗病原微生物定植等方面發(fā)揮著重要作用[1,2].得益于無菌小鼠的研究以及高通量測序技術(shù)、生物信息學等方法的不斷發(fā)展與普及應(yīng)用,腸道微生物群的復雜性及其對健康、疾病的影響逐漸被認識、確證,越來越多的腸道疾病被發(fā)現(xiàn)與腸道菌群相關(guān),對腸道疾病與腸道菌群關(guān)系的認識也逐漸由相關(guān)性報道深入到因果關(guān)系的研究[3].作為機體生物過程的重要協(xié)調(diào)者以及多種疾病發(fā)生發(fā)展過程中的共同參與者,加上其組成和功能的可塑性,腸道菌群逐漸成為一個極富吸引力的預防和治療疾病的靶標而引起研究者的高度關(guān)注.本文就腸道菌群與常見腸道疾病的相互關(guān)系以及治療腸道疾病的靶向腸道菌群策略進行概述,以期為腸道菌群研究和靶向菌群治療策略的應(yīng)用提供借鑒.

1 腸道菌群失調(diào)與胃腸疾病

人體腸道菌群主要由厚壁菌門、擬桿菌門、放線菌門和變形菌門四大類組成,其中厚壁菌門和擬桿菌門約占90%.微生物群落之間比例的巨大變化或新細菌群的擴張以及功能的改變導致宿主-微生物間相互作用的不平衡,稱為菌群失調(diào)[4].微生物組成或其衍生物（如代謝產(chǎn)物或基因毒素）的變化可影響宿主病生理信號通路,最終直接或間接地影響疾病的易感性,引起或參與腸道疾病,而這些成分也具有應(yīng)用于靶向治療的潛力.盡管菌群與一些疾病的因果關(guān)系尚不確切,但來自動物微生物群落移植模型的數(shù)據(jù)表明,其中一些變化不僅僅是疾病的后果[5,6](表1).

表1 與胃腸疾病相關(guān)的腸道菌群組成變化

1.1 腸易激綜合征 腸易激綜合征(irritable bowel syndrome,IBS)是一種常見的功能性胃腸疾病,典型癥狀是腹痛或不適,并與糞便性狀或頻率的改變有關(guān)[7].腸-腦軸、胃腸動力、腸道分泌、內(nèi)臟高敏感性、腸道通透性和免疫激活是IBS涉及的主要病理生理環(huán)節(jié),而研究發(fā)現(xiàn)這幾個環(huán)節(jié)都與腸道微生物群有關(guān)聯(lián)[8].此外,IBS的癥狀易受宿主遺傳、飲食、壓力和環(huán)境的影響,這些因素又都可以影響和塑造腸道菌群.

來自動物和人類研究的證據(jù)也支持腸道菌群在IBS的發(fā)展和遷延中的關(guān)鍵作用.首先,無菌小鼠模型提供了直接的證據(jù),將IBS-D患者的糞便菌群移植給無菌小鼠,可引起受體小鼠出現(xiàn)胃腸道轉(zhuǎn)運加快、低度炎癥和類似焦慮的腸道功能和行為改變,表明菌群失調(diào)可能是IBS的結(jié)腸運動功能障礙和行為癥狀的原因[5].然而,在沒有明確的IBS動物模型的情況下,直接的因果關(guān)系無法得到明確的證明.其次,感染后IBS (PI-IBS)的流行病學證據(jù)顯示,盡管清除了病原體,但胃腸道細菌感染導致持續(xù)功能性胃腸道癥狀的可能性增加且至少持續(xù)8年,有力的證明了腸道微生物群在部分患者的IBS發(fā)病中起著主要作用[9].最后,比對IBS患者和健康人群的臨床樣本也發(fā)現(xiàn),IBS患者伴隨著特定的腸道菌群及代謝特征.一項納入了24個病例對照研究的系統(tǒng)性回顧分析顯示,IBS人群的腸桿菌科(Enterobacteriaceae)、乳桿菌科(Lactobacillaceae)和擬桿菌屬(Bacteroides)增加,而不可培養(yǎng)的梭菌目Ⅰ(Clostridiales Ⅰ)、糞桿菌屬(Faecalibacterium) [包括普氏糞桿菌(Faecalibacterium prausnitzii)]和雙歧桿菌屬(Bifidobacterium)減少[10].一項縱向取樣的整合多組學方法的研究顯示,IBS-C患者中短鏈脂肪酸(short-chain fatty acids,SCFAs)和初級膽汁酸減少,而IBS-D患者中色胺和初級膽汁酸增多,研究人員表示這些菌群代謝物參與刺激結(jié)腸液體分泌,是造成IBS亞型癥狀的潛在原因[11].此外,IBS患者的特征菌群與其癥狀也有直接關(guān)聯(lián).IBS患者糞便菌群中Veillonella和Lactobacillus增加,而這兩類菌可產(chǎn)生乙酸、丙酸等有機酸,高乙酸和/或丙酸水平的IBS患者表現(xiàn)出更差的胃腸道癥狀、生活質(zhì)量和負面情緒[12];IBS-M和IBS-D患者糞便樣本中產(chǎn)丁酸鹽和甲烷的細菌豐度減少,而丁酸鹽已知能改善腸屏障結(jié)構(gòu)和功能,產(chǎn)甲烷的微生物是人體結(jié)腸中氫處理的主要機制,可以部分解釋IBS患者易出現(xiàn)腹脹的原因[13].

1.2 炎癥性腸病 炎癥性腸病(inflammatory bowel disease,IBD)包括潰瘍性結(jié)腸炎(ulcerative colitis,UC)和克羅恩病(Crohn’s disease,CD)兩大類.IBD被認為是由遺傳易感性、環(huán)境和微生物因素共同驅(qū)動的慢性免疫介導的腸道炎癥[14,15].

一方面,IBD患者中富集的微生物類群陸續(xù)被證實具有促炎癥的作用.Yilmaz等[16]通過重復兩項獨立的長期縱向IBD隊列之間的關(guān)鍵結(jié)果發(fā)現(xiàn),擬桿菌門、厚壁菌門和變形菌門是區(qū)別IBD患者和健康人群的主要微生物類群.此外,CD和UC患者分別具有特征性的細菌互作網(wǎng)絡(luò),包含毛螺菌科和瘤胃菌科的互作網(wǎng)絡(luò)的紊亂,與疾病的頻繁復發(fā)、抗TNF-α抗體治療低效化相關(guān)[16].Seishima等[17]發(fā)現(xiàn)UC和CD患者的微生物群在分類和功能上與健康人群有差異,糞腸球菌(E.faecium)作為差異最大的菌種與患者的疾病嚴重程度呈正相關(guān).相比于分離自健康人糞便中的屎腸球菌菌株,分離自UC患者糞便中的屎腸球菌菌株能促進IL-10敲除小鼠的結(jié)腸炎病理評分及炎癥因子表達升高[17].Chen等[18]發(fā)現(xiàn)具核梭桿菌(Fusobacterium nucleatum)富集于51.78%的UC組織中,并與UC的臨床病程、臨床活動度和難治性顯著相關(guān).進一步的機制研究顯示,具核梭桿菌可通過結(jié)腸上皮細胞中的NOD2而靶向上調(diào)CARD3,從而激活I(lǐng)L-17F/NF-κB信號通路,促進腸道炎癥的發(fā)生[18].活潑瘤胃球菌(Ruminococcus gnavus)是一種常見的厭氧革蘭氏陽性腸道細菌,在CD患者中活潑瘤胃球菌的相對豐度升高與癥狀增加相關(guān),研究發(fā)現(xiàn)R.gnavus可合成并分泌一種復雜的促炎癥性多糖,能以TLR4依賴性方式誘導樹突細胞分泌炎癥因子TNF-α[19].

另一方面,IBD患者中缺乏的微生物類群和細菌代謝物被發(fā)現(xiàn)具有抗炎作用.細菌代謝產(chǎn)物SCFAs可以調(diào)節(jié)保護性免疫并減輕組織炎癥.研究發(fā)現(xiàn),CD患兒腸道菌群中糞桿菌屬(Faecalibacterium)、羅氏菌屬(Roseburia)等SCFA產(chǎn)生菌減少,且糞便中的SCFA及非結(jié)合膽汁酸的水平降低,而英夫利昔單抗治療使CD患兒糞便中的膽鹽水解酶產(chǎn)生菌富集,結(jié)合膽汁酸水平降低、非結(jié)合膽汁酸水平和非結(jié)合/結(jié)合膽汁酸比例升高[20].在CD和UC患者中豐度顯著下降的羅氏菌屬(Roseburia)是存在于腸黏液層的乙酸-丁酸轉(zhuǎn)化器,能在黏液層產(chǎn)生抗炎作用[21].除SCFAs之外,具有調(diào)節(jié)宿主Treg細胞穩(wěn)態(tài)和TH17、Treg細胞分化作用的膽汁酸代謝在IBD患者中也被發(fā)現(xiàn)呈紊亂狀態(tài)[20,22].

1.3 結(jié)直腸腫瘤 腸癌是世界第三大常見的癌癥.對來自中國、奧地利、美國、德國和法國隊列的526個宏基因組樣本的聯(lián)合分析[23]顯示,結(jié)直腸癌患者的糞便菌群與健康人群存在明顯差異,并鑒定出脆弱擬桿菌(Bacteroides fragilis)、具核梭桿菌(Fusobacterium nucleatum)、Porphyromonas asaccharolytica、微小微單胞菌(Parvimonas micra)、中間普雷沃菌(Prevotella intermedia)、Alistipes finegoldii和Thermanaerovibrio acidaminovorans等7種在結(jié)直腸癌樣本中穩(wěn)定富集的、具有潛在診斷價值的細菌標志物,說明在腸癌的患者中存在菌群紊亂.針對多發(fā)性息肉樣腺瘤、粘膜內(nèi)癌和更晚期腸癌樣本的宏基因組學研究[24]發(fā)現(xiàn),在腸癌的多步發(fā)展階段存在微生物組的遷延轉(zhuǎn)移,具體表現(xiàn)為具核梭桿菌(Fusobacterium nucleatum spp.)的相對豐度從粘膜內(nèi)癌向更晚期病變持續(xù)升高以及在粘膜內(nèi)癌中共現(xiàn)的Atopobium parvulum和Actinomyces odontolyticus僅在多發(fā)性息肉樣腺瘤和/或粘膜內(nèi)癌中豐度顯著增加,表明菌群的變化發(fā)生在結(jié)直腸癌發(fā)展的早期階段,可能起到一個驅(qū)動作用或者是始動作用.將來自腸癌患者的糞便灌胃給無菌小鼠和抗生素處理后給予致癌物的小鼠,可增加兩種模型小鼠的息肉數(shù)量、腸道發(fā)育不良、增殖(Ki-67陽性)細胞水平、炎癥標記物(CXCR1、CXCR2、IL17A、IL22、IL23A)以及結(jié)腸中Th1和Th17細胞的比例[6],進一步說明了菌群可激活腸黏膜的免疫,誘發(fā)炎癥反應(yīng),促進上皮細胞增殖,從而起到促癌的作用.

對單菌功能的研究進一步論證了腸道微生物中的一類促致癌細菌具有引發(fā)和/或促進結(jié)腸癌的潛力.如研究發(fā)現(xiàn),脆弱擬桿菌(Bacteroides fragilis)產(chǎn)生的腸毒素是嬰幼兒腹瀉病的病因之一,亦可觸發(fā)結(jié)腸上皮中促致癌的多步驟炎癥級聯(lián)反應(yīng),與腸癌的發(fā)生發(fā)展密切相關(guān).脆弱擬桿菌毒素可引發(fā)結(jié)腸上皮細胞中的IL-17黏膜免疫信號,該信號又傳遞回上皮細胞激活NF-κb信號并觸發(fā)CXC趨化因子表達,繼而引發(fā)腫瘤前髓樣細胞浸潤至結(jié)腸遠端,與平行發(fā)生的Stat3信號共同促進產(chǎn)腸毒素脆弱擬桿菌介導的腫瘤發(fā)生[25].其他已鑒定的、與腸癌相關(guān)的促致癌菌還有攜帶pks的大腸桿菌(pks+ E.coli)[26]、具核梭桿菌(Fusobacterium nucleatum)[27]、厭氧消化鏈球菌(Peptostreptococcus anaerobius)[28]和產(chǎn)腸毒素的糞腸球菌.

2 靶向腸道菌群防治胃腸疾病的新策略

腸道菌群與宿主胃腸道之間的互作關(guān)系,以及其在胃腸疾病發(fā)生、發(fā)展中的作用,為預防和治療胃腸疾病提供了極具潛力的治療靶點(圖1).

圖1 潛在的預防和治療胃腸疾病的靶向菌群治療策略.

2.1 益生菌、益生元 益生菌是指當攝入足夠量時對宿主有健康益處的活的微生物[29].益生元是能夠被宿主體內(nèi)的菌群選擇性利用并轉(zhuǎn)化為有益于宿主健康的物質(zhì)[30].美國胃腸病協(xié)會建議,益生菌可考慮用于治療IBD的功能性癥狀[31].多項隨機、安慰劑對照臨床試驗也提供了積極的臨床證據(jù)支撐益生菌應(yīng)用于防治胃腸疾病[32-34].在一篇更新于JAMA臨床證據(jù)概要的Cochrane系統(tǒng)評價中,研究人員對來自15個國家的23項(N=3938)益生菌預防兒童抗生素相關(guān)性腹瀉的隨機試驗進行了評估,中等質(zhì)量的證據(jù)表明,預防性使用鼠李糖乳桿菌或布拉氏酵母菌與兒童(1個月至18歲)較低的抗生素相關(guān)性腹瀉發(fā)生率相關(guān),而不良事件也未見增加[35].納入12項隨機對照試驗(N=886)評估益生菌對UC作用的分析顯示,益生菌可顯著降低活動性UC患者的疾病活動指數(shù),而含有雙歧桿菌的益生菌可顯著緩解疾病,且維持緩解效果與美沙拉嗪無顯著差異[33].對200例IBD患者進行長達36個月以上的跟蹤調(diào)查,結(jié)果顯示服用益生菌可有效降低全身性類固醇用藥、住院及手術(shù)等不良事件的發(fā)生率,而且益生菌服用時間越長效果越好[36].

益生菌和益生元的作用機制復雜多樣,且通常有著菌株特異性.益生菌可以通過交叉喂養(yǎng)相互作用、降低Ph值改變胃腸道微環(huán)境、與致病菌競爭營養(yǎng)物質(zhì)和結(jié)合位點以及產(chǎn)生抗菌物質(zhì)如細菌素等途徑作用于宿主微生物群,從而發(fā)揮健康益處[37,38].此外,益生菌效應(yīng)分子(包括菌毛、脂磷壁酸、胞外多糖和各種表層蛋白質(zhì))可以直接與腸上皮、腸內(nèi)分泌和免疫細胞以及迷走神經(jīng)傳入纖維中的受體相互作用,產(chǎn)生增強腸道屏障完整性等局部腸道效應(yīng),以及通過宿主免疫、內(nèi)分泌、神經(jīng)系統(tǒng)介導的全身效應(yīng)[39,40].益生元效應(yīng)則通常是通過微生物群中特定群體對底物的消耗來介導的,從而促進它們的生長和代謝活性,同時也能通過交叉喂養(yǎng)相互作用促進生長等方式間接影響微生物群中的其他類群.益生元攝入引起的微生物組成和代謝物的變化會進一步影響宿主上皮、免疫、神經(jīng)和內(nèi)分泌信號,發(fā)揮對腸道功能、免疫反應(yīng)、抗炎、糖脂代謝、食欲和飽腹感等的調(diào)節(jié)[30,39].

雖然益生菌的應(yīng)用取得了一定范圍內(nèi)可喜的療效,但關(guān)于益生菌菌株和制劑的研究數(shù)據(jù)在一些情況下仍相互矛盾和有爭議,主要涉及益生菌的腸道定植、菌株水平的活性、與本地微生物群的相互作用、安全性和對宿主的影響等[41].

2.2 糞菌移植 糞菌移植(fecal microbiota transplantation,FMT)是將來自健康供體的糞便微生物群移植到患者的胃腸道,以糾正菌群失調(diào),重塑腸穩(wěn)態(tài)[42].FMT作為一種古老的腸道菌群干預措施,最早見于東晉葛洪所著《肘后備急方》,應(yīng)用糞便的懸浮液治療食物中毒和嚴重的腹瀉[43].與口服益生菌制劑相比,FMT具有移植菌群種類豐富、數(shù)量龐大、最大限度保留原有功能菌等優(yōu)勢.相較于利用某些特定的細菌來調(diào)控腸道菌群,FMT發(fā)揮作用的關(guān)鍵是依靠整體菌群來重建患者的內(nèi)穩(wěn)態(tài).

FMT被公認為是治療難治性難辨梭狀芽孢桿菌感染(Clostridium difficileinfections,CDI)的最有效方法,FMT一次治療CDI的有效率可達85%-90%,二次治療有效率可達100%,已被寫入美國CDI治療指南[44,45].除了CDI治療,FMT益處的最大證據(jù)來自對IBD患者的研究[46-48].Meta分析[48]顯示FMT治療CD患者緩解率為50.5%(42/83),明顯優(yōu)于UC患者[36%(201/555)].經(jīng)胃鏡至遠端十二指腸的FMT可顯著改善IBS患者的腹部癥狀、疲勞感和生活質(zhì)量,改善效果隨著劑量增加而增強[49].另有研究顯示,FMT移植方式的選擇對治療IBS的療效有直接影響,用腸鏡或鼻空腸管的方式移植新鮮或冷凍糞便或優(yōu)于糞菌膠囊[50].

盡管多項隨機對照研究證實了FMT的有效性,但其安全性和機制仍待深入研究,推進標準化FMT尚需更大努力.2019年6月,NEJM雜志曾報導了兩名免疫功能低下患者在接受來自同一供體的FMT后感染了具有超廣譜β-內(nèi)酰胺類藥物抗性的大腸桿菌,導致嚴重的菌血癥,其中一人死亡[51].為此,美國FDA發(fā)出關(guān)于FMT的安全性警示,要求在進行試驗性FMT時,必須針對供體的多重耐藥菌攜帶情況進行篩查和檢測,以減少耐藥菌傳播和感染風險[52].

2.3 噬菌體 噬菌體是以細菌為寄主的病毒,故也稱為細菌病毒.噬菌體能通過誘導細菌細胞裂解、影響細菌表型等機制塑造細菌群落[53].作為一種抗菌療法,相較于抗生素,噬菌體療法具有精準靶向的優(yōu)勢,可用于調(diào)節(jié)腸道菌群、殺滅多重耐藥菌[54].噬菌體療法在東歐部分國家被用作抗生素的替代或補充療法[55].在西方國家,噬菌體雖未被批準用于治療細菌感染,但噬菌體制劑已在美國被作為益生元(如前噬菌體、預噬菌體)在商業(yè)上可供人體使用[56].

在胃腸病中,噬菌體療法研究集中在感染性疾病上[56].幼兔模型顯示,單個噬菌體Phi_1可預防性和治療性地控制霍亂而沒有可檢測到的耐藥水平[57].黏附侵襲性大腸桿菌(adherent invasiveEscherichia coli,AIEC)是CD患者回腸黏膜中常見的致病菌,能與上皮細胞表面表達的CEACAM6受體結(jié)合,誘發(fā)結(jié)腸炎癥狀[58].以AIEC為靶標的三種噬菌體混合物可顯著減少CEABAC10轉(zhuǎn)基因小鼠(表達人CEACAM6 AIEC受體)腸黏膜中AIEC LF82菌株的定植,減輕DSS誘導的結(jié)腸炎癥狀[58],為靶向AIEC菌株治療CD提供了一種潛在的新選擇.

2.4 飲食干預 在影響宿主腸道菌群組成和結(jié)構(gòu)的因素中,飲食至關(guān)重要.飲食干預被認為是一種低成本、易操作的調(diào)節(jié)腸道菌群、治療菌群失調(diào)相關(guān)疾病的理想方式.在歐洲,純腸內(nèi)營養(yǎng)因具有更優(yōu)于皮質(zhì)類固醇的疾病緩解率和良好的安全性已被列為治療小兒CD的一線療法[59].多項隨機對照試驗顯示,低FODMAP飲食干預可充分緩解IBS患者的癥狀,同時伴有Bifidobacterium adolescentis、Bifidobacterium longum、Faecalibacterium prausnitzii、Bacteroides豐度的減少和糞便丁酸鹽水平的下降[60-62].在對緩解期或輕度UC患者進行的一項交叉試驗中[63],研究人員發(fā)現(xiàn),有針對性的攝入低脂飲食或高纖維飲食4 wk后,均可顯著改善生活質(zhì)量.此外,低脂飲食可顯著降低患者的炎癥標志物水平并改善失調(diào)的腸道菌群[63].基于IBD動物模型的研究發(fā)現(xiàn),水解蛋白飲食誘導的疾病緩解與致病菌(如E.coli、C.perfringens)的豐度降低、次級膽汁酸產(chǎn)生菌Clostridium hiranonis的豐度增加和次級膽汁酸(石膽酸、脫氧膽酸)水平的升高有關(guān)[64].

盡管事實上腸道菌群與胃腸道疾病的發(fā)生發(fā)展有關(guān),而且飲食可以用于緩解或治療胃腸疾病的癥狀,但飲食干預的多數(shù)機制尚不十分清楚.此外,基于腸道菌群的飲食干預,可因基線菌群的組成和功能的個體差異呈現(xiàn)不同療效[62].

3 結(jié)論

腸道菌群與疾病之間的關(guān)系不斷被挖掘,但仍面臨著不少挑戰(zhàn).一個亟待解決的問題是關(guān)于腸道微生物群與疾病之間的因果關(guān)系,這需要無菌動物、疾病模型和臨床證據(jù)的反復驗證.靶向腸道菌群的治療策略已展現(xiàn)出巨大潛力,盡管“精準”調(diào)節(jié)腸道菌群的最佳方式仍處于研究階段,但是毋容置疑的是,腸道微生物的調(diào)節(jié)將會更廣泛地用于整體健康改善和疾病輔助治療.通過進一步的研究,與疾病狀態(tài)有關(guān)的腸道菌群失調(diào)的真正的含義可以被更好的理解,以及什么是促進整體健康的最佳腸道菌群將會被更好地明確出來,這將對公共健康產(chǎn)生廣泛的積極作用.

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